The post-translationally modified variants of proteins such as glycoproteins, many of which are important for protein therapeutics and disease diagnostics, pose severe challenges to conventional 2D gels and capillary liquid chromatography because of their low abundance and distribution of structures. Developing a fast and accurate technology to characterize these proteins is an urgent need. Our Phase 1 results have demonstrated the feasibility of utilizing self-assembled nanoparticles, or colloidal crystals, as an ultrahigh resolution chromatographic medium, and the possibility of performing multilane chromatographic separation in a 2D microfluidic device. We propose to further these efforts to systematically achieve integration of the colloidal crystals into commercializable, fully functional devices with multilane sample injectors, and to demonstrate the level of improvement in the separation and detection of glycoproteins and their tryptic digests using both nanospray and matrix-assisted laser desorption and ionization (MALDI) mass spectrometry. The results of these proposed efforts, if successful, will be prototype products from consumables to systems that dramatically improve the toolbox available not only to practitioners interested in glycoproteins, but also in proteomics in general. The series of products will help transition the proteomics practitioners from their current work flow practice to entirely new device-based commercial products of superior performance and speed. The extent of glycosylation of proteins is broadly important in controlling signal and cell-cell recognition. Glycoproteins serve as biomarkers for disease, including many cancers. For hemoglobin, the extent of glycosylation is correlated with diabetes or prolonged stress. In a drug such as erythropoetin, a multibillion-dollar glycoprotein product used for treatment of various anemias and other conditions remains a major challenge, in the patients may die if the glycosylation in the dose given to them is not correct. The ability to analyze glycosylation levels of proteins, which is lacking, would allow advances in the understanding of this important process and the detection of biomarkers in clinical analysis. This proposal aims to create tools that will be effective and high speed for this analysis.